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Protein O-GIcNAcylation occurs in all animals and plants and is implicated in modulation of a wide range of cytosolic and nuclear protein functions, including gene silencing, nutrient and stress sensing, phosphorylation signaling, and diseases such as diabetes and Alzheimer's. The limiting factor impeding rapid progress in deciphering the biological functions of protein O-GIcNAcylation has been the inability to easily identify exact residues of modification. We describe a robust, high-sensitivity strategy able to assign O-GIcNAcylation sites of native modified peptides using electron transfer dissociation mass spectrometry. We have studied the murine postsynaptic density pseudoorganelle and report the assignment of 58 modification sites from a single experiment--significantly increasing the number of sites known in the literature. Components of several repressor complexes, such as NCoR1, polyhomeotic-like protein3, and EMSY, are modified. In addition, 28 O-GIcNAc sites were found on the protein Bassoon, effectively matching the number of phosphorylation sites reported previously on this protein. This finding suggests that on certain proteins, O-GIcNAcylation may be as extensive and important as phosphorylation in regulating protein function. Three of the newly discovered O-GIcNAc sites on Bassoon have previously been reported as phosphorylation sites, highlighting the interplay of the modifications. Surprisingly, several peptides with GIcNAc modifications on asparagines within the N-X-S/T consensus sequence were also observed from membrane protein extracellular domains. This powerful strategy fulfills a long-standing need in the biological community by facilitating modification site identifications that will accelerate understanding of the biological significance of this elusive regulatory posttranslational modification. modification site assignment | posttranslational modification | proteomics | Bassoon

Neuronal expression of growth-associated protein 43 (GAP-43) and the cell adhesion molecule L1 has been correlated with CNS axonal growth and regeneration, but it is not known whether expression of these molecules is necessary for axonal regeneration to occur. We have taken advantage of the fact that Purkinje cells do not express GAP-43 or L1 in adult mammals or regenerate axons into peripheral nerve grafts to test the importance of these molecules for axonal regeneration in vivo. Transgenic mice were generated in which Purkinje cells constitutively express L1 or both L1 and GAP-43 under the Purkinje cell-specific 17 promoter, and regeneration of Purkinje cell axons into peripheral nerve grafts implanted into the cerebellum was examined. Purkinje cells expressing GAP-43 or L1 showed minor enhancement of axonal sprouting. Purkinje cells expressing both GAP-43 and L1 showed more extensive axonal sprouting and axonal growth into the proximal portion of the graft. When a predegenerated nerve graft was implanted into double-transgenic mice, penetration of the graft by Purkinje cell axonal sprouts was strongly enhanced, and some axons grew along the entire intracerebral length of the graft (2.5-3.0 mm) and persisted for several months. The results demonstrate that GAP-43 and L1 coexpressed in Purkinje cells can act synergistically to switch these regeneration-incompetent CNS neurons into a regeneration-competent phenotype and show that coexpression of these molecules is a key regulator of the regenerative ability of intrinsic CNS neurons in vivo. axonal regeneration | CNS injury/repair | nerve graft | transgenic mice